Method for measuring blood components and biosensor and measuring instrument for use therein

ABSTRACT

The present invention provides a method of measuring a component in blood, by which the amounts of blood cells and an interfering substance can be measured with high accuracy and high reliability and the amount of the component can be corrected accurately based on the amounts of the blood cells and the interfering substance. In a sensor for measuring a blood component, a first working electrode  13  measures a current that flows during a redox reaction of a blood component, a second working electrode  17  measures the amount of blood cells, and a third working electrode  12  measures the amount of an interfering substance. Next, based on the measurement results, the amount of the blood component to be measured is corrected. Thus, more accurate and precise measurement of the amount of the blood component can be realized.

TECHNICAL FIELD

The present invention relates to a method of measuring a blood componentand to a biosensor and a measuring device used in the method.

BACKGROUND ART

Conventionally, sensors for measuring a blood component have been usedfor clinical tests, self-measurement of blood glucose level bydiabetics, etc. The configuration of the sensor for measuring a bloodcomponent is such that, for example, a cover is disposed on aninsulating substrate having a working electrode and a counter electrodeon its surface with a spacer intervening between the cover and theinsulating substrate. On the working electrode and the counterelectrode, a reagent containing an oxidoreductase, a mediator (anelectron carrier), and the like is provided, thereby forming an analysisportion. The analysis portion communicates with one end of a channel forleading blood to the analysis portion. The other end of the channel isopen toward the outside of the sensor so as to serve as a blood supplyport. Blood component analysis (e.g., analysis of blood glucose level)using the sensor configured as above is carried out in the followingmanner, for example. First, the sensor is set in a dedicated measuringdevice (a meter). Then, a fingertip or the like is punctured with alancet to cause bleeding, and the blood supply port of the sensor isbrought into contact with the blood that has come out. The blood isdrawn into the channel of the sensor by capillary action and flowsthrough the channel to be led to the analysis portion where the bloodcomes into contact with the reagent. Then, a redox reaction occursbetween a component in the blood and the oxidoreductase so thatelectrons move to the electrodes via the mediator. A current caused toflow at this time is detected, and the measuring device converts thedetected current into the amount of the blood component and displays thevalue obtained by the conversion.

However, the sensor response of an electrochemical blood glucose sensoras described above may be affected by an interfering substance such asan easily oxidizable compound (e.g., ascorbic acid or uric acid) and theamount of blood cells/hematocrit (Hct). Thus, in order to obtain anaccurate measured value, it is necessary to quantitate the interferingsubstance, the blood cells, or both the interfering substance and theblood cells and then correct the amount of the blood component (e.g.,the blood glucose level) based on the value(s) obtained by thequantitation. For example, there has been a sensor that corrects theamount of a blood component by measuring the amount of blood cells bythe use of two working electrodes and one reference electrode (seePatent Document 1). Other than this, there has been a method in whichthe amount of blood cells is measured using a mediator (see PatentDocument 2). Also, there has been a method in which an interferingsubstance is quantitated using an interfering substance-detectingelectrode (see Patent Document 3). However, the conventional techniqueshave a problem concerning the accuracy and the reliability of themeasured amounts of the blood cells and the interfering substance sothat the amount of the blood component cannot be corrected sufficiently.

[Patent Document 1] JP 2003-501627 A [Patent Document 2] Japanese PatentNo. 3369183 [Patent Document 3] Japanese Patent No. 3267933 DISCLOSUREOF INVENTION Problem to be Solved by the Invention

With the foregoing in mind, it is an object of the present invention toprovide a method of measuring a blood component, by which the amount ofa blood component can be corrected accurately by measuring the amount ofblood cells and the amount of an interfering substance with highaccuracy and high reliability and also to provide a sensor and ameasuring device used in the method.

Means for Solving Problem

In order to achieve the above object, the present invention provides amethod of measuring a component in blood, including the steps of:measuring a component in blood by causing a redox reaction between thecomponent and an oxidoreductase in the presence of a mediator, detectinga redox current caused by the redox reaction with a first electrodesystem including a working electrode and a counter electrode, andconverting a value of the detected current into an amount of thecomponent; correcting the amount of the component using an amount ofblood cells contained in the blood; and correcting the amount of thecomponent using an amount of an interfering substance contained in theblood. The correction step using the amount of the blood cells includes:providing a second electrode system including a working electrode and acounter electrode; providing a mediator on the counter electrode of thesecond electrode system but not on the working electrode of the secondelectrode system; supplying the blood to the second electrode system;applying a voltage to the second electrode system in this state to causea redox current to flow through the second electrode system; detectingthe redox current; converting a value of the detected redox current intothe amount of the blood cells; and correcting the amount of thecomponent based on the amount of the blood cells. The correction stepusing the amount of the interfering substance includes: providing athird electrode system including a working electrode and a counterelectrode; supplying the blood to the third electrode system; applying avoltage to the third electrode system in this state to cause a redoxcurrent to flow through the third electrode system; detecting the redoxcurrent; converting a value of the detected redox current into theamount of the interfering substance; and correcting the amount of thecomponent based on the amount of the interfering substance.

Furthermore, the present invention provides a biosensor for measuring acomponent in blood by causing a redox reaction of the component anddetecting a redox current caused by the redox reaction with anelectrode. The biosensor includes: a first analysis portion including afirst electrode system on which at least an oxidoreductase that actsupon the component and a mediator are provided; a second analysisportion including a second electrode system that includes a workingelectrode and a counter electrode, a mediator being provided on thecounter electrode but not on the working electrode; and a third analysisportion including a third electrode system that includes a workingelectrode and a counter electrode. In the first analysis portion, thecomponent in the blood is measured by causing a redox reaction betweenthe component and the oxidoreductase in the presence of the mediator anddetecting with the first electrode system a redox current caused to flowwhen a voltage is applied. In the second analysis portion, an amount ofblood cells contained in the blood is measured by supplying the blood tothe second electrode system, applying a voltage to the second electrodesystem in this state to cause a redox current to flow through the secondelectrode system, and detecting the redox current. In the third analysisportion, an amount of an interfering substance contained in the blood ismeasured by supplying the blood to the third electrode system, applyinga voltage to the third electrode system in this state to cause a currentto flow through the third electrode system, and detecting the current.

Still further, the present invention provides a measuring device formeasuring a component in blood using the above-described biosensor. Themeasuring device includes: measurement means for measuring a componentin blood by causing a redox reaction between the component and theoxidoreductase, detecting a redox current caused by the redox reactionwith the first electrode system, and converting the detected currentinto an amount of the component; correction means for correcting theamount of the component using an amount of blood cells contained in theblood; and correction means for correcting the amount of the componentusing an amount of an interfering substance contained in the blood. Thecorrection means using the amount of the blood cells uses the secondelectrode system for measuring the amount of the blood cells and carriesout the correction by applying a voltage to the second electrode systemin the presence of the blood to cause a current to flow, detecting thecurrent, converting a value of the detected current into the amount ofthe blood cells, and correcting the amount of the component based on theamount of the blood cells. The correction means using the amount of theinterfering substance uses the third electrode system for measuring theamount of the interfering substance and carries out the correction byapplying a voltage to the third electrode system in the presence of theblood to cause a current to flow, detecting the current, converting avalue of the detected current into the amount of the interferingsubstance, and correcting the amount of the components based on theamount of the interfering substance.

Effects of the Invention

As described above, in the measurement of a blood component, the amountof blood cells and the amount of an interfering substance can bemeasured with high accuracy by providing a plurality of workingelectrodes and measuring the amount of the blood component using one ofthe working electrodes and the amount of the blood cells and the amountof the interfering substance using the other working electrodes. As aresult, the correction of the amount of the blood component using theamounts of the blood cells and the interfering substance can beperformed with high accuracy and high reliability.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an exploded perspective view showing an example of a sensoraccording to the present invention.

FIG. 2 is a sectional view of the sensor shown in FIG. 1.

FIG. 3 is a plan view of the sensor shown in FIG. 1.

FIG. 4 is an exploded perspective view of another example of a sensoraccording to the present invention.

FIG. 5 is a sectional view of the sensor shown in FIG. 4.

FIG. 6 is a plan view of the sensor shown in FIG. 4.

FIG. 7 is a plan view of still another example of a sensor according tothe present invention.

FIG. 8 is an exploded perspective view showing still another example ofa sensor according to the present invention.

FIG. 9 is a sectional view of the sensor shown in FIG. 8.

FIG. 10 is a plan view of the sensor shown in FIG. 8.

FIG. 11 is a plan view of still another example of a sensor according tothe present invention.

FIG. 12 is a graph showing an example of the result of measurement of aresponse current for determining the amount of an interfering substance.

FIG. 13 is a graph showing an example of the result of measurement of aresponse current for determining the amount of blood cells.

FIG. 13A is a graph showing changes in response current (μA) over timeduring application of a voltage (V), and FIG. 13B is a graph showingchanges in difference in sensitivity (%) over time during theapplication of the voltage (V).

FIG. 14 is a graph showing another example of the result of measurementof a response current for determining the amount of blood cells. FIG.14A is a graph showing changes in response current (μA) over time duringapplication of a voltage (V), and FIG. 14B is a graph showing changes indifference in sensitivity (%) over time during the application of thevoltage (V).

FIG. 15 is a graph showing still another example of the result ofmeasurement of a response current for determining the amount of bloodcells. FIG. 15A is a graph showing changes in response current (μA) overtime during application of a voltage (V), and FIG. 15B is a graphshowing changes in difference in sensitivity (%) over time during theapplication of the voltage (V).

FIG. 16 is a graph showing still another example of the result ofmeasurement of a response current for determining the amount of bloodcells. FIG. 16A is a graph showing changes in response current (μA) overtime during application of a voltage (V), and FIG. 16B is a graphshowing changes in difference in sensitivity (%) over time during theapplication of the voltage (V).

FIG. 17 is a graph showing still another example of the result ofmeasurement of a response current for determining the amount of bloodcells. FIG. 17A is a graph showing changes in response current (μA) overtime during application of a voltage (V), and FIG. 17B is a graphshowing changes in difference in sensitivity (%) over time during theapplication of the voltage (V).

EXPLANATION OF REFERENCE NUMERALS

-   -   11 second counter electrode    -   12, 32, 52 third working electrode    -   13, 33, 53 first working electrode    -   14, 34, 54 liquid detecting electrode    -   15, 35, 55 first counter electrode    -   16, 36 third counter electrode    -   17, 37, 57 second working electrode    -   18, 19, 20, 39, 40, 60 round slit portion    -   21 second reagent layer    -   22, 42 third reagent layer    -   23, 43, 63 first reagent layer    -   24, 44, 64 channel    -   25, 45, 65 air hole    -   101, 301, 501 insulating substrate    -   102, 302, 502 spacer    -   103, 303, 503 cover

DESCRIPTION OF THE INVENTION

In the present invention, the correction based on the amount of theblood cells preferably is carried out using at least one of acalibration curve and a calibration table that have been preparedpreviously for showing the relationship between an amount of the bloodcells and an amount of the blood component. Furthermore, in the presentinvention, the correction based on the amount of the interferingsubstance preferably is carried out using at least one of a calibrationcurve and a calibration table that have been prepared previously forshowing the relationship between an amount of the interfering substanceand an amount of the blood component.

In the present invention, it is preferable that, in the third electrodesystem, a mediator is provided at least on the counter electrode.

In the present invention, at least one electrode selected from theworking electrodes and the counter electrodes of the first electrodesystem, the second electrode system, and the third electrode system mayserve also as any of the other electrodes. In the method of measuring ablood component according to the present invention, an electrode that isused as a working electrode in a certain step may be used as a counterelectrode in another step, and vice versa.

In the present invention, the order of measuring the amount of the bloodcomponent, the amount of the blood cells, and the amount of theinterfering substance is not particularly limited, but it is preferablethat the amount of the blood cells is measured last. Either of theamount of the blood component or the amount of the interfering substancemay be measured first, or they may be measured at the same time.

In the present invention, it is preferable that a voltage forpretreating the third electrode system is applied to the third electrodesystem before measuring the amount of the interfering substance. By thispretreatment, the surface of the third electrode system is cleaned, sothat the amount of the interfering substance and the amount of the bloodcells can be measured more accurately.

In the present invention, the voltage applied to the working electrodeof the third electrode system to perform the pretreatment preferably isin the range from 0.01 to 1 V relative to the counter electrode of thethird electrode system.

In the present invention, the voltage applied to the working electrodeof the third electrode system to measure the amount of the interferingsubstance preferably is in the range from 0.01 to 1 V and morepreferably in the range from 0.01 to 0.5 V relative to the counterelectrode of the third electrode system.

In the present invention, the voltage applied to the working electrodeof the second electrode system to measure the amount of the blood cellspreferably is at least 1 V, more preferably in the range from 1 to 10 V,and still more preferably in the range from 1 to 5 V relative to thecounter electrode of the second electrode system.

In the present invention, the blood component to be measured is, forexample, glucose, lactic acid, uric acid, bilirubin, cholesterol, or thelike. Furthermore, the oxidoreductase is selected as appropriatedepending on the blood component to be measured. Examples of theoxidoreductase include glucose oxidase, lactate oxidase, cholesteroloxidase, bilirubin oxidase, glucose dehydrogenase, and lactatedehydrogenase. The amount of the oxidoreductase is, for example, 0.01 to100 U, preferably 0.05 to 10 U, and more preferably 0.1 to 5 U per onesensor or one measurement. When the blood component to be measured isglucose, the oxidoreductase to be used preferably is glucose oxidase orglucose dehydrogenase.

Preferably, the biosensor according to the present invention isconfigured so that it further includes a channel for leading blood tothe biosensor, and the working electrode of the second analysis portionor the third analysis portion is located furthest upstream and theremaining electrodes are located downstream with respect to flow of theblood supplied from one end of the channel.

In the biosensor according to the present invention, it is preferablethat the first analysis portion is located furthest downstream in thechannel.

In the biosensor according to the present invention, it is not alwaysnecessary to provide a mediator on the working electrode of the thirdelectrode system. When the mediator is not provided on the workingelectrode of the third electrode system, the second electrode system andthe third electrode system may share the same working electrode. Also,in this case, either one or a combination of the electrodes of the firstelectrode system may be shared with at least one of the second electrodesystem and the third electrode system as the counter electrode.

In the biosensor according to the present invention, the secondelectrode system and the third electrode system may share an electrode,which is used as the counter electrode in the second electrode systemand as the working electrode in the third electrode system.

In the biosensor according to the present invention, the mediator may beprovided on the working electrode of the third electrode system, and inthis case, the second electrode system and the third electrode systemmay share an electrode, which is used as the working electrode in thethird electrode system and as the counter electrode in the secondelectrode system, and the third electrode system and the first electrodesystem may share the same counter electrode.

Preferably, the biosensor according to the present invention isconfigured so that it further includes a liquid detecting electrode, andthe liquid detecting electrode is located downstream from at least oneof the analysis portions so that whether or not the blood is supplied tothe at least one of the analysis portions can be detected with theliquid detecting electrode. The liquid detecting electrode can preventthe occurrence of measurement error due to the lack of blood supplied tothe biosensor, so that more accurate measurement of the amount of theblood component becomes possible. Note here that at least one of theworking electrodes and the counter electrodes of the first electrodesystem, the second electrode system, and the third electrode system mayserve also as the liquid detecting electrode. Furthermore, it ispreferable that the measuring device according to the present inventionfurther includes detection means that detects whether or not the bloodis supplied inside the biosensor with the liquid detecting electrode.

In the present invention, a mediator may be used. There is no particularlimitation regarding the mediator to be used. Examples of the mediatorinclude ferricyanides, p-benzoquinone, p-benzoquinone derivatives,phenazine methosulfate, methylene blue, ferrocene, and ferrocenederivatives. Among these, ferricyanides are preferable, and potassiumferricyanide is more preferable. The amount of the mediator to beblended is not particularly limited, but is, for example, 0.1 to 1000mM, preferably 1 to 500 mM, and more preferably 10 to 200 mM per onemeasurement or one sensor.

In the present invention, each of the electrodes preferably is coatedwith a polymeric material in order to prevent adhesion of impurities,oxidation of the electrode, and the like. Examples of the polymericmaterial include carboxymethyl cellulose (CMC), hydroxyethyl cellulose,hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethylhydroxyethyl cellulose, carboxyethyl cellulose, polyvinyl alcohol,polyvinylpyrrolidone, polyamino acid such as polylysine, polystyrenesulfonate, gelatin and derivatives thereof, polyacrylic acid and saltsthereof, polymethacrylic acid and salts thereof, starch and derivativesthereof, maleic anhydride polymer and salts thereof, and agarose gel andderivatives thereof. They may be used individually or two or more ofthem may be used together. The method of coating the electrode with apolymeric material is not particularly limited. For example, the coatingcan be achieved by providing a polymeric material solution, applying thesolution to the electrode surface, and then removing a solvent containedin the coating layer of the solution by drying.

Hereinafter, examples of a sensor for measuring a blood component andthe like according to the present invention will be described withreference to the drawings.

Example 1

FIGS. 1, 2, and 3 show one example of a sensor for measuring a bloodcomponent according to the present invention. FIG. 1 is an explodedperspective view of the sensor, FIG. 2 is a sectional view of thesensor, and FIG. 3 is a plan view of the sensor. In these threedrawings, the same components are given the same reference numerals.

As shown in the drawings, in this sensor, a first electrode systemincluding a first working electrode 13 and a first counter electrode 15,a second electrode system including a second working electrode 17 and asecond counter electrode 11, a third electrode system including a thirdworking electrode 12 and a third counter electrode 16, and a liquiddetecting electrode 14 are formed on an insulating substrate 101. Afirst reagent layer 23 is provided on the first electrode system, asecond reagent layer 21 is provided on the second counter electrode 11,and a third reagent layer 22 is provided on the third electrode system.The first reagent layer 23 contains an oxidoreductase such as glucosedehydrogenase and a mediator such as potassium ferricyanide andoptionally contains an enzyme stabilizer, a crystal homogenizing agent,and the like. Each of the second reagent layer 21 and the third reagentlayer 22 contains a mediator such as potassium ferricyanide andoptionally contains an enzyme stabilizer, a crystal homogenizing agent,and the like. A cover 103 is disposed on the insulating substrate 101 soas to cover an entire area excluding one end portion (the end portion onthe right in the drawings) with a spacer 102 intervening therebetween.In this sensor, the insulating substrate 101, the spacer 102, and thecover 103 form a channel 24 for leading blood to the respectiveelectrodes (11 to 17). The channel 24 extends to the other end portion(the end portion on the left in the drawings) of the sensor, and the tipof the channel 24 is open toward the outside of the sensor so as toserve as a blood supply port. The seven electrodes (11 to 17) areconnected to leads, respectively. These leads extend to theabove-described one end portion (the end portion on the right in thedrawings) of the sensor with the tip of each lead not being covered withthe cover but being exposed. The cover 103 has an air hole 25 at aportion corresponding to the right end portion of the channel 24.

In the present invention, the material of the insulating substrate isnot particularly limited, and may be, for example, polyethyleneterephthalate (PET), polycarbonate (PC), polyimide (PI), polyethylene(PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC),polyoxymethylene (POM), monomer-cast nylon (MC), polybutyleneterephthalate (PBT), a methacrylic resin (PMMA), an ABS resin (ABS), orglass. Among these, polyethylene terephthalate (PET), polycarbonate(PC), and polyimide (PI) are preferable, and polyethylene terephthalate(PET) is more preferable. The size of the insulating substrate is notparticularly limited. For example, the insulating substrate may have anoverall length of 5 to 100 mm, a width of 2 to 50 mm, and a thickness of0.05 to 2 mm; preferably an overall length of 7 to 50 mm, a width of 3to 20 mm, and a thickness of 0.1 to 1 mm; and more preferably an overalllength of 10 to 30 mm, a width of 3 to 10 mm, and a thickness of 0.1 to0.6 mm. Note here that the above description as to the material and thesize of the insulating substrate also applies to Examples 2 to 6 to bedescribed later.

The electrodes and the leads on the insulating substrate may be formedby, for example, forming a conductive layer with gold, platinum,palladium, or the like by sputtering or vapor deposition and thenprocessing the conductive layer into a particular electrode pattern witha laser. Examples of the laser include YAG lasers, CO₂ lasers, andexcimer lasers. Note here that this also applies to Examples 2 to 6 tobe described later.

The first reagent layer 23 is formed in the following manner. Forexample, an aqueous solution containing 0.1 to 5 U/sensor of glucosedehydrogenase, 10 to 200 mM of potassium ferricyanide, 1 to 50 mM ofmaltitol, and 20 to 200 mM of taurine is dropped on a round slit portion20 and then is dried. By providing this slit portion 20, it becomespossible to suppress the spreading of the droplet of the aqueoussolution, thereby allowing the first reagent layer 23 to be provided ata desired position more accurately. In this manner, the first reagentlayer 23 is formed on the first working electrode 13 and the firstcounter electrode 15. The drying may be natural drying or forced dryingusing warm air, for example. However, if the temperature of the warm airis too high, there is a possibility that the enzyme contained in thesolution might be deactivated. Thus, the temperature of the warm airpreferably is around 50° C.

The second reagent layer 21 is formed in the following manner. Forexample, an aqueous solution containing 10 to 200 mM of potassiumferricyanide and 20 to 200 mM of taurine is dropped on a round slitportion 18 and then is dried. By providing this slit portion 18, itbecomes possible to suppress the spreading of the droplet of the aqueoussolution, thereby allowing the second reagent layer 21 to be provided ata desired position more accurately. In this manner, the second reagentlayer 21 is formed on the second counter electrode 11.

The third reagent layer 22 is formed in the following manner. Forexample, an aqueous solution containing 10 to 200 mM of potassiumferricyanide and 20 to 200 mM of taurine is dropped on a round slitportion 19 and then is dried. By providing this slit portion 19, itbecomes possible to suppress the spreading of the droplet of the aqueoussolution, thereby allowing the third reagent layer 22 to be provided ata desired position more accurately. In this manner, the third reagentlayer 22 is formed on the third working electrode 12 and the thirdcounter electrode 16.

In the present invention, the material of the spacer is not particularlylimited. For example, the same material as that of the insulatingsubstrate can be used. The size of the spacer also is not particularlylimited. For example, the spacer may have an overall length of 5 to 100mm, a width of 2 to 50 mm, and a thickness of 0.01 to 1 mm; preferablyan overall length of 7 to 50 mm, a width of 3 to 20 mm, and a thickness0.05 to 0.5 mm; and more preferably an overall length of 10 to 30 mm, awidth of 3 to 10 mm, and a thickness of 0.05 to 0.25 mm. The spacer hasan I-shaped cut-away portion that serves as the channel for leadingblood. The cut-away portion may have, for example, an overall length of0.5 to 8 mm and a width of 0.1 to 5 mm; preferably an overall length of1 to 10 mm and a width of 0.2 to 3 mm; and more preferably an overalllength of 1 to 5 mm and a width of 0.5 to 2 mm. The cut-away portion maybe formed, for instance, by using a laser, a drill, or the like, or byforming the spacer using a die that can form the spacer provided withthe cut-away portion. Note here that the above description as to thematerial and the size of the spacer and the cut-away portion alsoapplies to Examples 2 to 6 to be described later.

In the present invention, the material of the cover is not particularlylimited. For example, the same material as that of the insulatingsubstrate can be used. It is more preferable that a portion of the covercorresponding to the ceiling of the channel for leading blood to thesensor is subjected to a treatment for imparting hydrophilicity. Thetreatment for imparting hydrophilicity may be carried out by, forexample, applying a surfactant or introducing a hydrophilic functionalgroup such as a hydroxyl group, a carbonyl group, or a carboxyl group tothe surface of the cover by plasma processing or the like. Furthermore,a layer formed of a surfactant such as lecithin may be formed on thereagent layer. The size of the cover is not particularly limited. Forexample, the cover may have an overall length of 5 to 100 mm, a width of3 to 50 mm, and a thickness of 0.01 to 0.5 mm; preferably an overalllength of 10 to 50 mm, a width of 3 to 20 mm, and a thickness of 0.05 to0.25 mm; and more preferably an overall length of 15 to 30 mm, a widthof 5 to 10 mm, and a thickness of 0.05 to 0.1 mm. The cover preferablyhas an air hole, and the shape of the air hole may be, for example,circular, oval, polygonal, or the like, and the maximum diameter thereofmay be, for example, 0.01 to 10 mm, preferably 0.05 to 5 mm, and morepreferably 0.1 to 2 mm. The air hole may be formed, for instance, byperforating the cover with a laser, a drill, or the like, or by formingthe cover using a die that can form the cover provided with the airhole. Note here that the above description as to the material and thesize of the cover and the air hole also applies to Examples 2 to 6 to bedescribed later.

By laminating the insulating substrate, the spacer, and the cover inthis order and integrating them, the sensor can be obtained. Theintegration can be achieved by adhering these three components with anadhesive or through heat-sealing. As the adhesive, an epoxy adhesive, anacrylic adhesive, a polyurethane adhesive, a thermosetting adhesive (ahot melt adhesive or the like), a UV curable adhesive, or the like canbe used, for example. Note here that this also applies to Examples 2 to6 to be described later.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1; Detecting Specimen (Blood))

The supply of blood to the sensor is detected by applying a voltagebetween the first counter electrode 15 and the liquid detectingelectrode 14. It is to be noted here that the combination of theelectrodes used for the blood supply detection is by no means limited tothe above combination. After the supply of the blood has been confirmed,the subsequent step is started. The voltage applied in Step 1 is, forexample, 0.05 to 1.0 V, preferably 0.1 to 0.8 V, and more preferably 0.2to 0.5 V.

(Step 2: Measuring Glucose)

After allowing glucose in the blood to react with an oxidoreductase fora certain period of time, a voltage is applied to the first workingelectrode 13. In this step, the first working electrode 13 is used as aworking electrode and the first counter electrode 15 is used as acounter electrode. A reduced mediator generated on the first workingelectrode 13 through the enzyme reaction is oxidized, and the oxidationcurrent caused at this time is detected. The glucose is allowed to reactwith the oxidoreductase for, for example, 0 to 60 seconds, preferably 1to 30 seconds, and more preferably 2 to 10 seconds. In Step 2, thevoltage applied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V,and more preferably 0.2 to 0.5 V, and the voltage application time is,for example, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 3: Measuring Amount of Interfering Substance)

By applying a voltage to the third working electrode 12, a currentcaused by the electrolytic oxidation reaction of the interferingsubstance is detected. In this step, the third working electrode 12 isused as a working electrode and the third counter electrode 16 is usedas a counter electrode. The amount of the interfering substance isdetermined based on the result of this detection. The amount of theinterfering substance is used for the correction in the measurement ofthe glucose. In this correction, the amount of the interfering substancedetermined using a previously prepared calibration curve showing therelationship between a current and an amount of the interferingsubstance may be used or alternatively the detected current may be usedas it is. In Step 3, the voltage applied is, for example, 0.01 to 1 Vand preferably 0.01 to 0.5 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. In the present example, both the workingelectrode and the counter electrode of the third electrode system areprovided with the mediator. Accordingly, a current caused by theelectrolytic oxidation reaction of the interfering substance is large,so that the amount of the interfering substance can be measured moreaccurately.

(Step 4: Measuring Amount of Blood Cells)

By applying a voltage to the second working electrode 17, anelectrolytic current depending on the amount of the blood cells can bedetected. In this step, the second working electrode 17 is used as aworking electrode and the second counter electrode 11 is used as acounter electrode. The amount of the blood cells is determined based onthe result of this detection. The amount of the blood cells is used forthe correction in the measurement of the glucose. In this correction,the amount of the blood cells determined using a previously preparedcalibration curve showing the relationship between an electrolyticcurrent and an amount of the blood cells may be used or alternativelythe detected electrolytic current may be used as it is. In Step 4, thevoltage applied is, for example, 1 to 10 V, preferably 1 to 5 V, andmore preferably 2 to 3 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds.

(Step 5: Correcting Amount of Blood Component)

The amount of the glucose obtained in Step 2 is corrected using theamount of the interfering substance measured in Step 3 and the amount ofthe blood cells measured in Step 4. Preferably, the correction iscarried out based on a calibration curve (including a calibration table)prepared previously. The corrected amount of the glucose is displayed onor stored in the measuring device.

Example 2

FIGS. 4, 5, and 6 show another example of a sensor for measuring a bloodcomponent according to the present invention. FIG. 4 is an explodedperspective view of the sensor, FIG. 5 is a sectional view of thesensor, and FIG. 6 is a plan view of the sensor. In these threedrawings, the same components are given the same reference numerals. Inthe sensor according to the present example, either one or thecombination of the electrodes of the first or third electrode systemserves as the counter electrode of the second electrode system of thesensor according to Example 1. Through the shared use of the electrodeas described above, it is possible to make the channel for leading bloodto the sensor shorter, thereby allowing the amount of blood required asa specimen to be reduced. Moreover, through the shared use of theelectrode, the number of the reagent layers can be reduced to two.

As shown in the drawings, in this sensor, a first electrode systemincluding a first working electrode 33 and a first counter electrode 35,a second working electrode 37, a third electrode system including athird working electrode 32 and a third counter electrode 36, and aliquid detecting electrode 34 are formed on an insulating substrate 301.A first reagent layer 43 is provided on the first electrode system, anda third reagent layer 42 is provided on the third electrode system. Thefirst reagent layer 43 contains an oxidoreductase such as glucosedehydrogenase and a mediator such as potassium ferricyanide andoptionally contains an enzyme stabilizer, a crystal homogenizing agent,and the like. The third reagent layer 42 contains a mediator such aspotassium ferricyanide and optionally contains an enzyme stabilizer, acrystal homogenizing agent, and the like. A cover 303 is disposed on theinsulating substrate 301 so as to cover an entire area excluding one endportion (the end portion on the right in the drawings) with a spacer 302intervening therebetween. In this sensor, the insulating substrate 301,the spacer 302, and the cover 303 form a channel 44 for leading blood tothe respective electrodes (32 to 37). The channel 44 extends to theother end portion (the end portion on the left in the drawings) of thesensor, and the tip of the channel 44 is open toward the outside of thesensor so as to serve as a blood supply port. The six electrodes (32 to37) are connected to leads, respectively. These leads extend to theabove-described one end portion (the end portion on the right in thedrawings) of the sensor with the tip of each lead not being covered withthe cover but being exposed. The cover 303 has an air hole 45 at aportion corresponding to the right end portion of the channel 44.

The first reagent layer 43 is formed in the following manner. Forexample, an aqueous solution containing 0.1 to 5 U/sensor of glucosedehydrogenase, 10 to 200 mM of potassium ferricyanide, 1 to 50 mM ofmaltitol, and 20 to 200 mM of taurine is dropped on a round slit portion40 and then is dried. By providing this slit portion 40, it becomespossible to suppress the spreading of the droplet of the aqueoussolution, thereby allowing the first reagent layer 43 to be provided ata desired position more accurately. In this manner, the first reagentlayer 43 is formed on the first working electrode 33 and the firstcounter electrode 35. The drying may be natural drying or forced dryingusing warm air, for example. However, if the temperature of the warm airis too high, there is a possibility that the enzyme contained in thesolution might be deactivated. Thus, the temperature of the warm airpreferably is around 50° C.

The third reagent layer 42 is formed in the following manner. Forexample, an aqueous solution containing 10 to 200 mM of potassiumferricyanide and 20 to 200 mM of taurine is dropped on a round slitportion 39 and then is dried. By providing this slit portion 39, itbecomes possible to suppress the spreading of the droplet of the aqueoussolution, thereby allowing the third reagent layer 42 to be provided ata desired position more accurately. In this manner, the third reagentlayer 42 is formed on the third working electrode 32 and the thirdcounter electrode 36.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1: Detecting Specimen (Blood))

The supply of blood to the sensor is detected by applying a voltagebetween the first counter electrode 35 and the liquid detectingelectrode 34. It is to be noted here that the combination of theelectrodes used for the blood supply detection is by no means limited tothe above combination. After the supply of the blood has been confirmed,the subsequent step is started. The voltage applied in Step 1 is, forexample, 0.05 to 1.0 V, preferably 0.1 to 0.8 V, and more preferably 0.2to 0.5 V.

(Step 2: Measuring Glucose)

After allowing glucose in the blood to react with an oxidoreductase fora certain period of time, a voltage is applied to the first workingelectrode 33. In this step, the first working electrode 33 is used as aworking electrode and the first counter electrode 35 is used as acounter electrode. A reduced mediator generated on the first workingelectrode 33 through the enzyme reaction is oxidized, and the oxidationcurrent caused at this time is detected. The glucose is allowed to reactwith the oxidoreductase for, for example, 0 to 60 seconds, preferably 1to 30 seconds, and more preferably 2 to 10 seconds. In Step 2, thevoltage applied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V,and more preferably 0.2 to 0.5 V, and the voltage application time is,for example, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 3: Measuring Amount of Interfering Substance)

By applying a voltage to the third working electrode 32, a currentcaused by the electrolytic oxidation reaction of the interferingsubstance is detected. In this step, the third working electrode 32 isused as a working electrode and the third counter electrode 36 is usedas a counter electrode. The amount of the interfering substance isdetermined based on the result of this detection. The amount of theinterfering substance is used for the correction in the measurement ofthe glucose. In this correction, the amount of the interfering substancedetermined using a previously prepared calibration curve showing therelationship between a current and an amount of the interferingsubstance may be used or alternatively the detected current may be usedas it is. In Step 3, the voltage applied is, for example, 0.01 to 1 Vand preferably 0.01 to 0.5 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds.

(Step 4: Measuring Amount of Blood Cells)

By applying a voltage to the second working electrode 37, anelectrolytic current depending on the amount of the blood cells can bedetected. In this step, the second working electrode 37 is used as aworking electrode and the third working electrode 32 is used as acounter electrode. The amount of the blood cells is determined based onthe result of this detection. The amount of the blood cells is used forthe correction in the measurement of the glucose. In this correction,the amount of the blood cells determined using a previously preparedcalibration curve showing the relationship between an electrolyticcurrent and an amount of the blood cells may be used or alternativelythe detected electrolytic current may be used as it is. In Step 4, thevoltage applied is, for example, 1 to 10 V, preferably 1 to 5 V, andmore preferably 2 to 3 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Preferably, Step 4 is performed as a laststep in the series of steps. Although the third working electrode 32 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstworking electrode 33 alone, the first counter electrode 35 alone, thethird counter electrode 36 alone, the combination of the third workingelectrode 32 and the third counter electrode 36, or the combination ofthe first working electrode 33 and the first counter electrode 35 alsomay be used as the counter electrode.

The reason why the measurement of the amount of the blood cells isperformed last is as follows. When the amount of the blood cells ismeasured before measuring the amount of the blood component and theamount of the interfering substance, the following phenomenon occurs.That is, although the mediator that initially is in an oxidized state(e.g., potassium ferricyanide) is provided on the electrode(s) used asthe counter electrode, the mediator that is in a reduced state (e.g.,potassium ferrocyanide) is generated by the measurement of the amount ofthe blood cells. If the amount of the blood component and the amount ofthe interfering substance are measured thereafter, the reduced mediatorthus generated causes a background noise, resulting in an error in themeasured value.

(Step 5: Correcting Amount of Blood Component)

The amount of the glucose obtained in Step 2 is corrected using theamount of the interfering substance measured in Step 3 and the amount ofthe blood cells measured in Step 4. Preferably, the correction iscarried out based on a calibration curve (including a calibration table)prepared previously. The corrected amount of the glucose is displayed onor stored in the measuring device.

Example 3

FIG. 7 shows still another example of a sensor for measuring a bloodcomponent according to the present invention. FIG. 7 is a plan viewshowing an electrode pattern in this sensor, which corresponds to theelectrode pattern shown in FIG. 6 in which either one or the combinationof the electrodes of the first electrode system is shared with the thirdelectrode system as the counter electrode. Except for the above, thissensor has the same configuration as the sensor according to Example 2,and the components, the configuration of the reagent layers, productionmethod, etc. of this sensor are the same as those of the sensoraccording to Example 2.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1: Detecting Specimen (Blood))

The supply of blood to the sensor is detected by applying a voltagebetween the first counter electrode 35 and the liquid detectingelectrode 34. It is to be noted here that the combination of theelectrodes used for the blood supply detection is by no means limited tothe above combination. After the supply of the blood has been confirmed,the subsequent step is started. The voltage applied in Step 1 is, forexample, 0.05 to 1.0 V, preferably 0.1 to 0.8 V, and more preferably 0.2to 0.5 V.

(Step 2: Measuring Glucose)

After allowing glucose in the blood to react with an oxidoreductase fora certain period of time, a voltage is applied to the first workingelectrode 33. In this step, the first working electrode 33 is used as aworking electrode and the first counter electrode 35 is used as acounter electrode. A reduced mediator generated on the first workingelectrode 33 through the enzyme reaction is oxidized, and the oxidationcurrent caused at this time is detected. The glucose is allowed to reactwith the oxidoreductase for, for example, 0 to 60 seconds, preferably 1to 30 seconds, and more preferably 2 to 10 seconds. In Step 2, thevoltage applied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V,and more preferably 0.2 to 0.5 V, and the voltage application time is,for example, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 3: Measuring Amount of Interfering Substance)

By applying a voltage to the third working electrode 32, a currentcaused by the electrolytic oxidation reaction of the interferingsubstance is detected. In this step, the third working electrode 32 isused as a working electrode and the first working electrode 33 is usedas a counter electrode. The amount of the interfering substance isdetermined based on the result of this detection. The amount of theinterfering substance is used for the correction in the measurement ofthe glucose. In this correction, the amount of the interfering substancedetermined using a previously prepared calibration curve showing therelationship between a current and an amount of the interferingsubstance may be used or alternatively the detected current may be usedas it is. In Step 3, the voltage applied is, for example, 0.01 to 1 Vand preferably 0.01 to 0.5 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Although the first working electrode 33 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstcounter electrode 35 alone or the combination of the first workingelectrode 33 and the first counter electrode 35 also may be used as thecounter electrode.

When the first working electrode 33 or the combination of the firstworking electrode 33 and the first counter electrode 35 is used as thecounter electrode, Step 3 preferably is performed after the amount ofthe blood component has been measured. The reason why the amount of theinterfering substance is measured after the amount of the bloodcomponent has been measured is as follows. When the amount of theinterfering substance is measured before measuring the amount of theblood component, the following phenomenon occurs. That is, although themediator that initially is in an oxidized state (e.g., potassiumferricyanide) is provided on the electrode(s) used as the counterelectrode, the mediator that is in a reduced state (e.g., potassiumferrocyanide) is generated by the measurement of the amount of theinterfering substance. If the reduced mediator thus generated diffuseson the first working electrode 33 for measuring the amount of the bloodcomponent, the mediator causes a background noise during the measurementof the amount of the blood component, resulting in an error in themeasured value.

However, when the first counter electrode 35 alone is used as thecounter electrode, Step 3 may be performed before measuring the amountof the blood component. The reason for this is that the amount of themediator in a reduced state (e.g., potassium ferrocyanide) generated onthe first counter electrode 35 is not large enough to diffuse on thefirst working electrode 33 and thus there is little chance that it mightcause a background noise.

(Step 4: Measuring Amount of Blood Cells)

By applying a voltage to the second working electrode 37, anelectrolytic current depending on the amount of the blood cells can bedetected. In this step, the second working electrode 37 is used as aworking electrode and the third working electrode 32 is used as acounter electrode. The amount of the blood cells is determined based onthe result of this detection. The amount of the blood cells is used forthe correction in the measurement of the glucose. In this correction,the amount of the blood cells determined using a previously preparedcalibration curve showing the relationship between an electrolyticcurrent and an amount of the blood cells may be used or alternativelythe detected electrolytic current may be used as it is. In Step 4, thevoltage applied is, for example, 1 to 10 V, preferably 1 to 5 V, andmore preferably 2 to 3 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Preferably, Step 4 is performed as a laststep in the series of steps. Although the third working electrode 32 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstworking electrode 33 alone, the first counter electrode 35 alone, or thecombination of the first working electrode 33 and the first counterelectrode 35 also may be used as the counter electrode.

The reason why the measurement of the amount of the blood cells isperformed last is the same as that described in Example 2.

(Step 5: Correcting Amount of Blood Component)

The amount of the glucose obtained in Step 2 is corrected using theamount of the interfering substance measured in Step 3 and the amount ofthe blood cells measured in Step 4. Preferably, the correction iscarried out based on a calibration curve (including a calibration table)prepared previously. The corrected amount of the glucose is displayed onor stored in the measuring device.

Example 4

FIGS. 8, 9, and 10 show still another example of a sensor for measuringa blood component according to the present invention. FIG. 8 is anexploded perspective view of the sensor, FIG. 9 is a sectional view ofthe sensor, and FIG. 10 is a plan view of the sensor. In these threedrawings, the same components are given the same reference numerals. Thesensor according to the present example corresponds to the sensoraccording to Example 3 from which the third reagent layer provided onthe third working electrode is removed. As shown in the drawings, inthis sensor, a first electrode system including a first workingelectrode 53 and a first counter electrode 55, a second workingelectrode 57, a third working electrode 52, and a liquid detectingelectrode 54 are formed on an insulating substrate 501. A first reagentlayer 63 is provided on the first electrode system. The first reagentlayer 63 contains an oxidoreductase such as glucose dehydrogenase and amediator such as potassium ferricyanide and optionally contains anenzyme stabilizer, a crystal homogenizing agent, and the like. A cover503 is disposed on the insulating substrate 501 so as to cover an entirearea excluding one end portion (the end portion on the right in thedrawings) with a spacer 502 intervening therebetween. In this sensor,the insulating substrate 501, the spacer 502, and the cover 503 form achannel 64 for leading blood to the respective electrodes (52 to 55, and57). The channel 64 extends to the other end portion (the end portion onthe left in the drawings) of the sensor, and the tip of the channel 64is open toward the outside of the sensor so as to serve as a bloodsupply port. The five electrodes (52 to 55, and 57) are connected toleads, respectively. These leads extend to the above-described one endportion (the end portion on the right in the drawings) of the sensorwith the tip of each lead not being covered with the cover but beingexposed. The cover 503 has an air hole 65 at a portion corresponding tothe right end portion of the channel 64.

The first reagent layer 63 is formed in the following manner. Forexample, an aqueous solution containing 0.1 to 5 U/sensor of glucosedehydrogenase, 10 to 200 mM of potassium ferricyanide, 1 to 50 mM ofmaltitol, and 20 to 200 mM of taurine is dropped on a round slit portion60 and then is dried. By providing this slit portion 60, it becomespossible to suppress the spreading of the droplet of the aqueoussolution, thereby allowing the first reagent layer 63 to be provided ata desired position more accurately. In this manner, the first reagentlayer 63 is formed on the first working electrode 53 and the firstcounter electrode 55. The drying may be natural drying or forced dryingusing warm air, for example. However, if the temperature of the warm airis too high, there is a possibility that the enzyme contained in thesolution might be deactivated. Thus, the temperature of the warm airpreferably is around 50° C.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1: Detecting Specimen (Blood))

The supply of blood to the sensor is detected by applying a voltagebetween the first counter electrode 55 and the liquid detectingelectrode 54. It is to be noted here that the combination of theelectrodes used for the blood supply detection is by no means limited tothe above combination. After the supply of the blood has been confirmed,the subsequent step is started. The voltage applied in Step 1 is, forexample, 0.05 to 1.0 V, preferably 0.1 to 0.8 V, and more preferably 0.2to 0.5 V.

(Step 2: Measuring Glucose)

After allowing glucose in the blood to react with an oxidoreductase fora certain period of time, a voltage is applied to the first workingelectrode 53. In this step, the first working electrode 53 is used as aworking electrode and the first counter electrode 55 is used as acounter electrode. A reduced mediator generated on the first workingelectrode 53 through the enzyme reaction is oxidized, and the oxidationcurrent caused at this time is detected. The glucose is allowed to reactwith the oxidoreductase for, for example, 0 to 60 seconds, preferably 1to 30 seconds, and more preferably 2 to 10 seconds. In Step 2, thevoltage applied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V,and more preferably 0.2 to 0.5 V, and the voltage application time is,for example, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 3: Measuring Amount of Interfering Substance)

By applying a voltage to the third working electrode 52, a currentcaused by the electrolytic oxidation reaction of the interferingsubstance is detected. In this step, the third working electrode 52 isused as a working electrode and the first working electrode 53 is usedas a counter electrode. The amount of the interfering substance isdetermined based on the result of this detection. The amount of theinterfering substance is used for the correction in the measurement ofthe glucose. In this correction, the amount of the interfering substancedetermined using a previously prepared calibration curve showing therelationship between a current and an amount of the interferingsubstance may be used or alternatively the detected current may be usedas it is. In Step 3, the voltage applied is, for example, 0.01 to 1 Vand preferably 0.01 to 0.5 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Although the first working electrode 53 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstcounter electrode 55 alone or the combination of the first workingelectrode 53 and the first counter electrode 55 also may be used as thecounter electrode.

When the first working electrode 53 or the combination of the firstworking electrode 53 and the first counter electrode 55 is used as thecounter electrode, Step 3 preferably is performed after the amount ofthe blood component has been measured. The reason why the amount of theinterfering substance is measured after the amount of the bloodcomponent has been measured is the same as that described in Example 3.

(Step 4: Measuring Amount of Blood Cells)

By applying a voltage to the second working electrode 57, anelectrolytic current depending on the amount of the blood cells can bedetected. In this step, the second working electrode 57 is used as aworking electrode and the first working electrode 53 is used as acounter electrode. The amount of the blood cells is determined based onthe result of this detection. The reason why the first working electrode53 is used as the counter electrode is as follows. After the amount ofthe blood component has been measured, the mediator that is in anoxidized state (e.g., potassium ferricyanide) is present dominantly onthe first working electrode 53. Thus, when the first working electrode53 is used as the counter electrode for measuring the amount of theblood cells, it is possible to suppress the electrolytic reductionreaction occurring at the counter electrode from being arate-determining step. The amount of the blood cells is used for thecorrection in the measurement of the glucose. In this correction, theamount of the blood cells determined using a previously preparedcalibration curve showing the relationship between an electrolyticcurrent and an amount of the blood cells may be used or alternativelythe detected electrolytic current may be used as it is. In Step 4, thevoltage applied is, for example, 1 to 10 V, preferably 1 to 5 V, andmore preferably 2 to 3 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Preferably, Step 4 is performed as a laststep in the series of steps. Although the first working electrode 53 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstcounter electrode 55 alone or the combination of the first workingelectrode 53 and the first counter electrode 55 also may be used as thecounter electrode.

The reason why the measurement of the amount of the blood cells isperformed last is the same as that described in Example 2.

(Step 5: Correcting Amount of Blood Component)

The amount of the glucose obtained in Step 2 is corrected using theamount of the interfering substance measured in Step 3 and the amount ofthe blood cells measured in Step 4. Preferably, the correction iscarried out based on a calibration curve (including a calibration table)prepared previously. The corrected amount of the glucose is displayed onor stored in the measuring device.

Example 5

FIG. 11 shows still another example of a sensor for measuring a bloodcomponent according to the present invention. FIG. 11 is a plan viewshowing an electrode pattern in this sensor, which corresponds to theelectrode pattern shown in FIG. 10 in which the second working electrodeserves also as the third working electrode. Except for the above, thissensor has the same configuration as the sensor according to Example 4,and the components, the configuration of the reagent layers, productionmethod, etc. of this sensor are the same as those of the sensoraccording to Example 4.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1: Detecting Specimen (Blood))

The supply of blood to the sensor is detected by applying a voltagebetween the first counter electrode 55 and the liquid detectingelectrode 54. It is to be noted here that the combination of theelectrodes used for the blood supply detection is by no means limited tothe above combination. After the supply of the blood has been confirmed,the subsequent step is started. The voltage applied in Step 1 is, forexample, 0.05 to 1.0 V, preferably 0.1 to 0.8 V, and more preferably 0.2to 0.5 V.

(Step 2: Measuring Glucose)

After allowing glucose in the blood to react with an oxidoreductase fora certain period of time, a voltage is applied to the first workingelectrode 53. In this step, the first working electrode 53 is used as aworking electrode and the first counter electrode 55 is used as acounter electrode. A reduced mediator generated on the first workingelectrode 53 through the enzyme reaction is oxidized, and the oxidationcurrent caused at this time is detected. The glucose is allowed to reactwith the oxidoreductase for, for example, 0 to 60 seconds, preferably 1to 30 seconds, and more preferably 2 to 10 seconds. In Step 2, thevoltage applied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V,and more preferably 0.2 to 0.5 V, and the voltage application time is,for example, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 3: Measuring Amount of Interfering Substance)

By applying a voltage to the second working electrode 57, a currentcaused by the electrolytic oxidation reaction of the interferingsubstance is detected. In this step, the second working electrode 57 isused as a working electrode and the first working electrode 53 is usedas a counter electrode. The amount of the interfering substance isdetermined based on the result of this detection. The amount of theinterfering substance is used for the correction in the measurement ofthe glucose. In this correction, the amount of the interfering substancedetermined using a previously prepared calibration curve showing therelationship between a current and an amount of the interferingsubstance may be used or alternatively the detected current may be usedas it is. In Step 3, the voltage applied is, for example, 0.01 to 1 Vand preferably 0.01 to 0.5 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Although the first working electrode 53 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstcounter electrode 55 alone or the combination of the first workingelectrode 53 and the first counter electrode 55 also may be used as thecounter electrode.

When the first working electrode 53 or the combination of the firstworking electrode 53 and the first counter electrode 55 is used as thecounter electrode, Step 3 preferably is performed after the amount ofthe blood component has been measured. The reason why the amount of theinterfering substance is measured after the amount of the bloodcomponent has been measured is the same as that described in Example 3.

(Step 4: Measuring Amount of Blood Cells)

By applying a voltage to the second working electrode 57, anelectrolytic current depending on the amount of the blood cells can bedetected. In this step, the second working electrode 57 is used as aworking electrode and the first working electrode 53 is used as acounter electrode. The amount of the blood cells is determined based onthe result of this detection. The reason why the first working electrode53 is used as the counter electrode is the same as that described inExample 4. The amount of the blood cells is used for the correction inthe measurement of the glucose. In this correction, the amount of theblood cells determined using a previously prepared calibration curveshowing the relationship between an electrolytic current and an amountof the blood cells may be used or alternatively the detectedelectrolytic current may be used as it is. In Step 4, the voltageapplied is, for example, 1 to 10 V, preferably 1 to 5 V, and morepreferably 2 to 3 V, and the voltage application time is, for example,0.001 to 60 seconds, preferably 0.01 to 10 seconds, and more preferably0.01 to 5 seconds. Preferably, Step 4 is performed as a last step in theseries of steps. Although the first working electrode 53 is used as thecounter electrode in the present example, the present invention is notlimited thereto. It should be noted that the first counter electrode 55alone or the combination of the first working electrode 53 and the firstcounter electrode 55 also may be used as the counter electrode.

The reason why the measurement of the amount of the blood cells isperformed last is the same as that described in Example 2.

(Step 5: Correcting Amount of Blood Component)

The amount of the glucose obtained in Step 2 is corrected using theamount of the interfering substance measured in Step 3 and the amount ofthe blood cells measured in Step 4. Preferably, the correction iscarried out based on a calibration curve (including a calibration table)prepared previously. The corrected amount of the glucose is displayed onor stored in the measuring device.

Example 6

Example 6 is directed to an example where a sensor as shown in FIG. 11was used as in Example 5 and an electrode pretreatment further isperformed.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1: Detecting Specimen (Blood))

The supply of blood to the sensor is detected by applying a voltagebetween the first counter electrode 55 and the liquid detectingelectrode 54. It is to be noted here that the combination of theelectrodes used for the blood supply detection is by no means limited tothe above combination. After the supply of the blood has been confirmed,the subsequent step is started. The voltage applied in Step 1 is, forexample, 0.05 to 1.0 V, preferably 0.1 to 0.8 V, and more preferably 0.2to 0.5 V.

(Step 2: Pretreating Electrode)

A voltage is applied to the second working electrode 57 to clean thesurface of the second working electrode 57. In this step, the secondworking electrode 57 is used as a working electrode and the firstcounter electrode 55 is used as a counter electrode. In Step 2, thevoltage applied preferably is in the range from 0.01 to 1 V and morepreferably from 0.01 to 0.5 V, and the voltage application time is, forexample, 0.001 to 30 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. By performing this pretreatment, thesurface of the second working electrode 57 is cleaned, so that theamount of the interfering substance can be measured more accurately.Step 2 may be performed simultaneously with or after Step 4 (themeasurement of glucose) to be described later.

As long as Step 2 is performed before measuring the amount of theinterfering substance and the amount of the blood cells, Step 2 can beperformed at the most effective timing from the viewpoint of simplicityin operation and reduction in time required for the whole measurementprocess.

(Step 3: Measuring Amount of Interfering Substance)

By applying a voltage to the second working electrode 57, a currentcaused by the electrolytic oxidation reaction of the interferingsubstance is detected. In this step, the second working electrode 57 isused as a working electrode and the first counter electrode 55 is usedas a counter electrode. The amount of the interfering substance isdetermined based on the result of this detection. The amount of theinterfering substance is used for the correction in the measurement ofthe glucose. In this correction, the amount of the interfering substancedetermined using a previously prepared calibration curve showing therelationship between a current and an amount of the interferingsubstance may be used or alternatively the detected current may be usedas it is. In Step 3, the voltage applied is, for example, 0.01 to 1 Vand preferably 0.01 to 0.5 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Although the first counter electrode 55 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstworking electrode 53 alone or the combination of the first workingelectrode 53 and the first counter electrode 55 also may be used as thecounter electrode.

When the first working electrode 53 or the combination of the firstworking electrode 53 and the first counter electrode 55 is used as thecounter electrode, Step 3 preferably is performed after the amount ofthe blood component has been measured. The reason why the amount of theinterfering substance is measured after the amount of the bloodcomponent has been measured is the same as that described in Example 3.However, when the first counter electrode 55 alone is used as thecounter electrode as in the present Example 6, Step 3 may be performedbefore measuring the amount of the blood component. The reason for thisis that, in this case, the amount of the mediator in a reduced state(e.g., potassium ferrocyanide) generated on the first counter electrode55 is not large enough to diffuse on the first working electrode 33 andthus there is little chance that it might cause a background noise.

(Step 4: Measuring Glucose)

After allowing glucose in the blood to react with an oxidoreductase fora certain period of time, a voltage is applied to the first workingelectrode 53. In this step, the first working electrode 53 is used as aworking electrode and the first counter electrode 55 is used as acounter electrode. A reduced mediator generated on the first workingelectrode 53 through the enzyme reaction is oxidized, and the oxidationcurrent caused at this time is detected. The glucose is allowed to reactwith the oxidoreductase for, for example, 0 to 60 seconds, preferably 1to 30 seconds, and more preferably 2 to 10 seconds. In Step 2, thevoltage applied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V,and more preferably 0.2 to 0.5 V, and the voltage application time is,for example, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 5: Measuring Amount of Blood Cells)

By applying a voltage to the second working electrode 57, anelectrolytic current depending on the amount of the blood cells can bedetected. In this step, the second working electrode 57 is used as aworking electrode and the first working electrode 53 is used as acounter electrode. The amount of the blood cells is determined based onthe result of this detection. The amount of the blood cells is used forthe correction in the measurement of the glucose. In this correction,the amount of the blood cells determined using a previously preparedcalibration curve showing the relationship between an electrolyticcurrent and an amount of the blood cells may be used or alternativelythe detected electrolytic current may be used as it is. In Step 4, thevoltage applied is, for example, 1 to 10 V, preferably 1 to 5 V, andmore preferably 2 to 3 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. Preferably, Step 4 is performed as a laststep in the series of steps. Although the first working electrode 53 isused as the counter electrode in the present example, the presentinvention is not limited thereto. It should be noted that the firstcounter electrode 55 alone or the combination of the first workingelectrode 53 and the first counter electrode 55 also may be used as thecounter electrode.

The reason why the measurement of the amount of the blood cells isperformed last is the same as that described in Example 2.

(Step 6: Correcting Amount of Blood Component)

The amount of the glucose obtained in Step 4 is corrected using theamount of the interfering substance measured in Step 3 and the amount ofthe blood cells measured in Step 5. Preferably, the correction iscarried out based on a calibration curve (including a calibration table)prepared previously. The corrected amount of the glucose is displayed onor stored in the measuring device.

As an example of the blood component measurement, Examples 1 to 6describe the case where the glucose concentration in blood is measured.However, the present invention is by no means limited thereto. Asalready described above, the present invention also is useful for themeasurement of other blood components, such as lactic acid andcholesterol.

Although several electrode patterns are shown in Examples 1 to 6, thepresent invention is by no means limited thereto. It is to be noted herethat the electrode pattern can be changed as appropriate depending onthe purpose or the conditions of use of the sensor, for example.

Reference Example 1

A sensor having a configuration shown in FIGS. 1, 2, and 3 was produced.The first reagent layer 23 was formed by dissolving glucosedehydrogenase (1 to 5 U), potassium ferricyanide (60 mM), and taurine(80 mM) in a CMC aqueous solution (0.1 wt %) to prepare a reagentsolution, dropping the reagent solution on the round slit portion 20,and then drying it. The second reagent layer 21 and the third reagentlayer 22 were formed by dissolving potassium ferricyanide (60 mM) andtaurine (80 mM) in a CMC aqueous solution (0.1 wt %) to prepare areagent solution, dropping the reagent solution on the round slitportions 18 and 19, and then drying it.

Using this sensor, a response current for determining the amount of aninterfering substance was measured. Ascorbic acid was used as an exampleof an easily oxidizable interfering substance, and blood samplesrespectively containing 0, 5, 10, and 20 mg/dl of ascorbic acid wereprovided. Using the thus-prepared four blood samples, a current flowingthrough the third electrode system was measured. The measurement wasperformed by applying a voltage of 0.5 V to the third working electrode12 for 3 seconds.

Next, using the same sensor, a response current for determining theamount of blood cells was measured. Three types of blood samples inwhich the amounts of blood cells were adjusted to be 25%, 45%, and 65%,respectively, were provided. Using the thus-prepared three bloodsamples, an electrolytic current flowing through the second electrodesystem was measured. Using the third working electrode 32 as a counterelectrode, the measurement was performed by applying a voltage of 2.5 Vto the second working electrode 17 for 3 seconds.

Reference Example 2

A sensor having a configuration shown in FIGS. 4, 5, and 6 was produced.The first reagent layer 43 was formed by dissolving glucosedehydrogenase (1 to 5 U), potassium ferricyanide (60 mM), and taurine(80 mM) in a CMC aqueous solution (0.1 wt %) to prepare a reagentsolution, dropping the reagent solution on the round slit portion 40,and then drying it. The third reagent layer 42 was formed by dissolvingpotassium ferricyanide (60 mM) and taurine (80 mM) in a CMC aqueoussolution (0.1 wt %) to prepare a reagent solution, dropping the reagentsolution on the round slit portion 39, and then drying it.

Using this sensor, a response current for determining the amount of aninterfering substance was measured. Ascorbic acid was used as an exampleof an easily oxidizable interfering substance, and blood samplesrespectively containing 0, 5, 10, and 20 mg/dl of ascorbic acid wereprovided. Using the thus-prepared four blood samples, a current flowingthrough the third electrode system was measured. The measurement wasperformed by applying a voltage of 0.5 V to the third working electrode32 for 3 seconds.

Next, using the same sensor, a response current for determining theamount of blood cells was measured. Three types of blood samples inwhich the amounts of blood cells were adjusted to be 25%, 45%, and 65%,respectively, were provided. Using the thus-prepared three bloodsamples, an electrolytic current flowing through the second electrodesystem was measured. The measurement was performed by applying a voltageof 2.5 V to the second working electrode 37 for 3 seconds.

Reference Example 3

A sensor having a configuration shown in FIG. 7 was produced. The firstreagent layer 43 was formed by dissolving glucose dehydrogenase (1 to 5U), potassium ferricyanide (60 mM), and taurine (80 mM) in a CMC aqueoussolution (0.1 wt %) to prepare a reagent solution, dropping the reagentsolution on the round slit portion 40, and then drying it. The thirdreagent layer 42 was formed by dissolving potassium ferricyanide (60 mM)and taurine (80 mM) in a CMC aqueous solution (0.1 wt %) to prepare areagent solution, dropping the reagent solution on the round slitportion 39, and then drying it.

Using this sensor, a response current for determining the amount of aninterfering substance was measured. Ascorbic acid was used as an exampleof an easily oxidizable interfering substance, and blood samplesrespectively containing 0, 5, 10, and 20 mg/dl of ascorbic acid wereprovided. Using the thus-prepared four blood samples, a current flowingthrough the third electrode system was measured. Using the first workingelectrode 33 as a counter electrode, the measurement was performed byapplying a voltage of 0.5 V to the third working electrode 32 for 3seconds.

Next, using the same sensor, a response current for determining theamount of blood cells was measured. Three types of blood samples inwhich the amounts of blood cells were adjusted to be 25%, 45%, and 65%,respectively, were provided. Using the thus-prepared three bloodsamples, an electrolytic current flowing through the second electrodesystem was measured. Using the third working electrode 32 as a counterelectrode, the measurement was performed by applying a voltage of 2.5 Vto the second working electrode 37 for 3 seconds.

Reference Example 4

A sensor having a configuration shown in FIGS. 8, 9, and 10 wasproduced. The first reagent layer 63 was formed by dissolving glucosedehydrogenase (1 to 5 U), potassium ferricyanide (60 mM), and taurine(80 mM) in a CMC aqueous solution (0.1 wt %) to prepare a reagentsolution, dropping the reagent solution on the round slit portion 60,and then drying it. Using this sensor, a response current fordetermining the amount of an interfering substance was measured.Ascorbic acid was used as an example of an easily oxidizable interferingsubstance, and blood samples respectively containing 0, 5, 10, and 20mg/dl of ascorbic acid were provided. Using the thus-prepared four bloodsamples, a current flowing through the third electrode system wasmeasured. Using the first working electrode 53 as a counter electrode,the measurement was performed by applying a voltage of 0.5 V to thethird working electrode 52 for 3 seconds.

Next, using the same sensor, a response current for determining theamount of blood cells was measured. Three types of blood samples inwhich the amounts of blood cells were adjusted to be 25%, 45%, and 65%,respectively, were provided. Using the thus-prepared three bloodsamples, an electrolytic current flowing through the second electrodesystem was measured. Using the first working electrode 53 as a counterelectrode, the measurement was performed by applying a voltage of 2.5 Vto the second working electrode 57 for 3 seconds.

Reference Example 5

A sensor having a configuration shown in FIG. 11 was produced. The firstreagent layer 63 was formed by dissolving glucose dehydrogenase (1 to 5U), potassium ferricyanide (60 mM), and taurine (80 mM) in a CMC aqueoussolution (0.1 wt %) to prepare a reagent solution, dropping the reagentsolution on the round slit portion 60, and then drying it.

Using this sensor, a response current for determining the amount of aninterfering substance was measured. Ascorbic acid was used as an exampleof an easily oxidizable interfering substance, and blood samplesrespectively containing 0, 5, 10, and 20 mg/dl of ascorbic acid wereprovided. Using the thus-prepared four blood samples, a current flowingthrough the third electrode system was measured. Using the first workingelectrode 53 as a counter electrode, the measurement was performed byapplying a voltage of 0.5 V to the third working electrode 52 for 3seconds.

Next, using the same sensor, a response current for determining theamount of blood cells was measured. Three types of blood samples inwhich the amounts of blood cells were adjusted to be 25%, 45%, and 65%,respectively, were provided. Using the thus-prepared three bloodsamples, an electrolytic current flowing through the second electrodesystem was measured. Using the first working electrode 53 as a counterelectrode, the measurement was performed by applying a voltage of 2.5 Vto the second working electrode 57 for 3 seconds.

FIG. 12 is a graph showing the result of measurements of the responsecurrents for determining the amount of the interfering substance inReference Examples 1 to 5. As can be seen from FIG. 12, the responsecurrents reflecting the amounts of the interfering substance could bedetected.

FIGS. 13 to 17 show the result of measurements of the response currentsfor determining the amount of the blood cells in Reference Examples 1 to5. In FIGS. 13 to 17, FIGS. 13A to 17A are graphs each showing changesin response current (μA) over time during the application of the voltage(V), and FIGS. 13B to 17B are graphs each showing changes in differencein sensitivity (%) over time during the application of the voltage (V).As can be seen from these drawings, according to the sensors ofReference Examples 1 to 5, the difference in sensitivity did not dependon the voltage application time, so that the response current reflectingthe amount of the blood cells could be detected definitely.

INDUSTRIAL APPLICABILITY

The method of measuring a blood component according to the presentinvention measures the amounts of an interfering substance and bloodcells with high accuracy and high reliability and corrects the amount ofthe blood component based on the amounts of the interfering substanceand the blood cells. Thus, the method of the present invention canmeasure the blood component with high accuracy and high reliability.Accordingly, the present invention is useful for the measurement of ablood component such as glucose.

1-25. (canceled)
 26. A method of measuring a glucose in blood by using abiosensor, the biosensor comprising: an electrode system comprising anelectrode on which a reagent is disposed and an electrode on which noreagent is disposed, said reagent including an oxidoreductase that actsupon the glucose and a mediator, the method comprising: a first step ofobtaining a first current value by applying a first voltage to theelectrode system, said first current value including an effect of aninterfering substance and an effect of blood cells or hematocrit (Hct),a second step of obtaining a second current value by applying a secondvoltage to the electrode system, said second current value determiningthe effect of blood cells or hematocrit (Hct), and thereafter, a thirdstep of obtaining a corrected glucose value by using the first currentvalue and the second current value.
 27. The method of measuring aglucose in blood by using a biosensor according to claim 26, wherein thefirst voltage is applied for a time of 0.01 to 5 seconds.
 28. The methodof measuring a glucose in blood by using a biosensor according to claim26, wherein the second voltage is applied for a time of 0.01 to 5seconds.
 29. The method of measuring a glucose in blood by using abiosensor according to claim 26, wherein the first step is performedbefore the second step.